Vibrio fischeri: The Lux Operon
Vibrio fischeri is a species of bacteria found in the ocean which have the ability to emit light. These bacteria are found in the light-producing glands of the Hawaiian bobtail squid and the pinecone fish, Monocentris japonicus. The bacteria live symbiotically with the squid and fish, and produce light to presumably help the animal blend in with the light coming from the ocean above. But what produces that light in the bacteria? The answer is simple. An enzyme called luciferase, which creates light after the oxidization of a flavin and aldehyde, is responsible for bioluminescence in bacteria like V. fischeri ''(1 ). The genetics behind this particular trait are not quite as simple, and a lot of work was done by Engerbrecht and colleagues to find the answer to how this process is actually encoded in the bacterial genome. '' fish.jpg|Pinecone fish, Monocentris japonicus (from endlessblue.jp) Hawaiian_bobtail_squid04.jpg|Hawaiian bobtail squid (from news.wisc.edu) VF labs-medmicro-wisc-edu.jpg|''Vibrio fischeri (from labs.medmicro.wisc.edu) vibrio.jpg|SEM of Vibrio fischeri (from visualsunlimited.photoshelter.com) '' Lux Operon While the regulation of the lux operon, the set of genes responsible for the production of the correct enzymes needed to drive the luciferase reaction, was discovered in the the previous year byt the same group, Engerbrecht and colleauges published the discovery of the genes and gene functions of the lux operon in 1984 (1 , 2 ). The lux operon is bidirectional and encodes seven genes (luxR, I, C, D, A, B, ''and ''E) needed for bioluminescence. The luxI gene encodes the autoinducer, which is secreted by the bacterium and enters a neighboring cell and induces the transcription of the operon. The luxR gene encodes the protein that responds to the autoinducer. Three genes, luxC, D, ''and ''E, are involved in the production of the aldehyde substrate. Finally, luxA ''and ''B encode for the alpha and beta subunits of the luciferase enzyme (1 ). The number, identity, and function of these genes were discovered through extensive genetic analyses. Decoding the Lux Operon: Genetic Analysis The work to discover the genetic elements responsible for bioluminescence began with the development of mutant bacteria that produced little or no light. The bacteria used were E. coli transformed with a plasmid containing the lux operon, which was previously isolated. E. coli ''containing mutants in the lux operon were generated (or mutagenized) through incubation with hydroxylamine. There were four main phenotypes generated; mutants that were unable to produce or respond to exogenous autoinducer, mutants that were unable to produce autoinducer but could respond (by producing light) when the autoinducer was introduced, mutants that produced light when exogenous aldehyde was introduced (indicating that these mutants could not produce aldehyde, but could respond to it), and mutants that could not respond to the introduction of exogenous aldehyde (indicating that these mutants could neither produce, nor respond to, aldehyde) (1 ). Now that mutations in the lux operon were made, the next step in the process was to do a complementation analysis. Another set of mutations were made in the wild-type lux operon using the Tn5 transposon. The insertion of the transposon had previously been mapped in the lux operon DNA, allowing the researchers to map phenotypes to particular regions of the lux operon. Complementation is used to determine the number of genes involved in a particular phenotype. The assay works by transforming bacteria with two plasmids, each with a mutation in the region of DNA being examined, which is the lux operon in this study. Both mutations cause a lack of bioluminescence. If the two mutations are in different genes, then the phenotype will be restored. The restoration occurs because the cell now has two copies of each gene and if two different genes have a mutation causing a lack of function, there is another "healthy" gene that will produce the correct phenotype. Non-complementation, seen when there is no restoration in phenotype, occurs when the two mutations are in the same gene. Seven groups of non-complementary mutations were seen in the lux operon, suggesting the lux operon contains seven genes, referred to as ''luxR, I, D, C, E, A, ''and ''B (1 ). Engerbrecht and colleagues also determined the molecular weight of each of the gene products of hte lux operon. To do this, they used minicells, which are essentially miniature cells but contain only RNA and have the ability to produce protein. These minicells were made from the E. coli containing the plasmids with the wild type lux operon and the operons with each of the genes knocked out by creating mutants with a chain-terminating sequence in each gene. The gene products were separated on a polyacrylamide gel. Since the mutations were mapped to particular genes, the molecular weights were determined based on which proteins were absent in each mutant when compared to the wild type. The molecular weights for the gene products of luxR, I, C, D, E, A, ''and ''B were found to be 27 kDa, 25 kDa, 53 kDa, 33 kDa, 42 kDa, 40 kDa, and 38 kDa, respectively (1 ). This analysis of the lux operon has been extremely important in the development of other studies into bioluminescence and in the use of the operon in various molecular biology studies. References 1. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC345387/ Engebrecht, J. and Silverman, M. Identification of genes and gene products necessary for bacterial bioluminescence. Proc. Natl. Acad. Sci. (1984)] 2. [http://www.ncbi.nlm.nih.gov/pubmed/6831560 Engebrecht, J. et al. Bacterial Bioluminescence: isolation and genetic analysis of functions from Vibrio Fischeri. Cell. (1983)]